背景:肺癌仍然是全球癌症相关死亡率的主要原因。尽管最近的治疗进展提供了有针对性的治疗方法,耐药性和全身毒性的发展仍然是人们关注的主要问题.细胞外囊泡(EV),特别是那些来自间充质基质细胞(MSC),作为有前途的药物递送系统获得了关注,提供生物相容性和最小的免疫反应。认识到常规2D细胞培养系统在模拟肿瘤微环境方面的局限性,本研究旨在描述在肺癌和正常肺组织中使用患者特异性类器官模型的原理验证方法,以及在个性化医疗方法中使用源自诱导多能干细胞(iPSC)-MSC的自体EVs的可行性.
方法:首先,我们将健康的成纤维细胞重新编程为iPSC。接下来,我们将患者来源的iPSC分化为分支肺类器官(BLO),并从患者来源的肿瘤组织中产生患者匹配的肺癌类器官(LCO).我们展示了从iPSC分化MSC和从iPSC-MSC分离EV的简化过程,用0.07µg/mL的细胞毒性剂顺铂封装,并应用于两个类器官模型。用LDH和CCK8试验记录顺铂和负载顺铂的EV的细胞毒性。
结果:成纤维细胞来源的iPSC显示正常核型,多能性染色,和三系分化。iPSC来源的BLO显示肺标志物的表达,如TMPRSS2和MUC5A,而患者匹配的LCO显示Napsin和CK5的表达。接下来,我们比较了在肺癌类器官模型和健康肺类器官模型中,负载顺铂的iPSC-MSC来源的EV与空EV和单用顺铂的效果.不出所料,当LCO用20µg/mL顺铂治疗时,我们发现细胞毒性作用.用空EV治疗LCO和BLO在24小时后产生细胞毒性作用。然而,装载0.07µg/mL顺铂的EV在两个类器官模型中均未诱导任何细胞毒性作用。
结论:我们报告了未来使用自体或同种异体iPSC-MSCEVs作为肺癌药物递送试验的原理证明。然而,由于时间和劳动力密集的过程,我们得出的结论是,目前这种管道对于个性化方法可能不可行。
BACKGROUND: Lung cancer remains a leading cause of cancer-related mortality globally. Although recent therapeutic advancements have provided targeted treatment approaches, the development of resistance and systemic toxicity remain primary concerns. Extracellular vesicles (EVs), especially those derived from mesenchymal stromal cells (MSC), have gained attention as promising drug delivery systems, offering biocompatibility and minimal immune responses. Recognizing the limitations of conventional 2D cell culture systems in mimicking the tumor microenvironment, this study aims to describe a proof-of-principle approach for using patient-specific organoid models for both lung cancer and normal lung tissue and the feasibility of employing autologous EVs derived from induced pluripotent stem cell (
iPSC)-MSC in personalized medicine approaches.
METHODS: First, we reprogrammed healthy fibroblasts into
iPSC. Next, we differentiated patient-derived iPSC into branching lung organoids (BLO) and generated patient-matched lung cancer organoids (LCO) from patient-derived tumor tissue. We show a streamlined process of MSC differentiation from
iPSC and EV isolation from
iPSC-MSC, encapsulated with 0.07 µg/mL of cytotoxic agent cisplatin and applied to both organoid models. Cytotoxicity of cisplatin and cisplatin-loaded EVs was recorded with LDH and CCK8 tests.
RESULTS: Fibroblast-derived
iPSC showed a normal karyotype, pluripotency staining, and trilineage differentiation. iPSC-derived BLO showed expression of lung markers, like TMPRSS2 and MUC5A while patient-matched LCO showed expression of Napsin and CK5. Next, we compared the effects of
iPSC-MSC derived EVs loaded with cisplatin against empty EVs and cisplatin alone in lung cancer organoid and healthy lung organoid models. As expected, we found a cytotoxic effect when LCO were treated with 20 µg/mL cisplatin. Treatment of LCO and BLO with empty EVs resulted in a cytotoxic effect after 24 h. However, EVs loaded with 0.07 µg/mL cisplatin failed to induce any cytotoxic effect in both organoid models.
CONCLUSIONS: We report on a proof-of-principle pipeline towards using autologous or allogeneic iPSC-MSC EVs as drug delivery tests for lung cancer in future. However, due to the time and labor-intensive processes, we conclude that this pipeline might not be feasible for personalized approaches at the moment.